Affinity chromatographic purification of antibodies to a biotinylated fusion protein expressed in Escherichia coli.
HOP1, a protein component of the synaptonemal complex in Saccharomyces cerevisiae which is believed to play an important role in meiotic synapsis, was expressed in Escherichia coli as a fusion protein incorporating a "tag" polypeptide which is biotinylated naturally in the bacteria. The HOP1 fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinant protein was used to immunize rabbits and produce polyclonal antibodies. Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward. Affinity-purified antibodies show high binding capacity to HOP1 recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction. The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction.
de Dios Alché
J
J
Plant Sciences Department, University of Oxford, South Parks Road, Oxford, OX1 3RB, United Kingdom.
Dickinson
H
H
D000818:Animals; D000908:Antibodies, Fungal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001710:Biotin; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015870:Gene Expression; D007074:Immunoglobulin G / Q000302:isolation & purification; D008540:Meiosis / Q000235:genetics; D008970:Molecular Weight; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility
D000908:Antibodies, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C086672:HOP1 protein, S cerevisiae; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001710:Biotin